A major objective of the proposed research is to define the growth requirements and metabolism of virulent Treponema pallidum, the causative agent of syphilis, to enable its cultivation in vitro with retained virulence. This information may help to identify treponemal virulence factors and suggest ways of interrupting the host-treponeme interaction. Virulent treponemes are extracted from infected rabbit tissue and incubated in assay medium containing specific metabolic inhibitors and radioactive substrates to measure selective incorporation and utilization of radiolabeled precursors by treponemes. By modifying the basic assay procedure, protein and nucleic acid synthesis, carbohydrate metabolism, and intermediate and terminal energy-generating pathways of virulent treponemes will be examined. In addition, cell-free extracts of virulent treponemes purified by velocity sedimentation in diatrizoate will be analyzed for enzymic activity and cytochrome and flavoprotein content. In other studies, cocultivation of virulent treponemes in a variety of animal cell cultures will be attempted. Also, various enzymes which are either lacking in treponemes or have diminished activity will be added to culture media to see whether their presence improves treponemal survival or stimulates metabolic activity.